A transgenic approach to live imaging of heparan sulfate modification patterns

Abstract

Heparan sulfate (HS) glycosaminoglycan chains contain highly modified HS domains that are separated by sections of sparse or no modification. HS domains are central to the role of HS in protein binding and mediating protein-protein interactions in the extracellular matrix. Since HS domains are not genetically encoded, they are impossible to visualize and study with conventional methods in vivo. Here we describe a transgenic approach using previously described single chain variable fragment (scFv) antibodies that bind HS in vitro and on tissue sections with different specificities. By engineering a secretion signal and a fluorescent protein to the scFvs and transgenically expressing these fluorescently tagged antibodies in Caenorhabditis elegans, we are able to directly visualize specific HS domains in live animals (Attreed et al. Nat Methods 9(5):477-479, 2012). The approach allows concomitant colabeling of multiple epitopes, the study of HS dynamics and, could lend itself to a genetic analysis of HS domain biosynthesis or to visualize other nongenetically encoded or posttranslational modifications.

Figure 1

a. Schematic of the basic HS structure organized into domains with varying degrees of sulfation. Modifications and putative growth factor binding sites are indicated. b. Schematic of the secreted scFv antibody fusion (inset) and the basic plasmid construct (1). The construct was designed in a modular fashion to allow for easy swapping of the heavy chains, targeting sequence, 3’ UTR, fluorophores, and promoters. Two-headed arrows indicate alternative modules that could be exchanged with existing modules. Common restriction sites are indicated. NLS: nuclear localization signal. c. Diagram of a transgenic worm expressing the antibody construct from specific cells (coelomocytes in this example). The antibody is secreted into the pseudocoelom where it diffuses and binds to heparan sulfate domains. Unbound antibody is taken up by the coelomocytes which absorb material from the pseudocoelom (9).

Figure 2

a.-b. Epifluorescent micrographs of transgenic animals expressing an EW4G1::GFP (a) or LKIV69::GFP fusion (b) under control of the coelomocyte-specific punc-122 (23) promoter show staining of the nervous system in C. elegans. In all panels, filled or open green arrow heads indicate the dorsal and ventral nerve cords, respectively and, magenta arrowheads indicate neuronal staining associated with the C. elegans nerve ring. No comparable staining was observed when a control scFv antibody fusion (MPB49::GFP) was expressed under the same conditions (1). The gut with characteristic vesicular autofluorescence is indicated. Scale bars indicate 25 μm in all panels and p: pharynx. c. Epifluorescent micrographs of transgenic animals expressing an HS3A8::GFP antibody fusion under control of the coelomocyte-specific punc-122 (upper panel) and a presynaptic marker (mCherry::RAB-3 fusion) under control of a promoter specific for GABAergic neurons (middle panel)(Attreed & Bülow, unpublished). A merged image (lower panel) shows partial colocalization of HS3A8-specific HS epitopes with the presynaptic marker in GABAergic neurons. d. Epifluorescent micrographs of transgenic animals expressing an HS4C3::DsRed2 antibody fusion under control of the coelomocyte-specific punc-122 (upper panel) and a presynaptic marker (SNB-1::GFP) under control of a promoter specific for GABAergic neurons (middle panel)(juIs1, (20)). A merged image (lower panel) shows partial colocalization of HS4C3-specific HS epitopes with the presynaptic marker in GABAergic neurons.